Mutated von willebrand factor

ABSTRACT

The present invention provides a modified polypeptide which binds Factor VIII. The modified polypeptide comprises a sequence as shown in SEQ ID NO:3 in which the sequence comprises at least one modification at a position selected from the group consisting of L18, V42, K149, N248, S279, V320, T325, Q395 and K418 such that the modified polypeptide binds to Factor VIII with an off rate lower than a reference polypeptide comprising an unmodified SEQ ID NO:3.

This application is the United States national stage entry under 35 U.S.C. § 371 of International Application No. PCT/AU2017/050009, filed on Jan. 6, 2017 and published as WO 2017/117630 A9, which claims priority to Australian Patent Application No. 2016900033, filed on Jan. 7, 2016. The contents of these applications are each incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to polypeptides, in particular modified von Willebrand Factor which exhibits improved binding affinity to Factor VIII. The invention further relates to a complex comprising the polypeptide and FVIII, to a polynucleotide encoding the polypeptide of the invention and a method of producing the polypeptide. Furthermore, the invention concerns the therapeutic or prophylactic use of the polypeptide or complex of the invention for treating bleeding disorders.

BACKGROUND OF THE INVENTION

There are various bleeding disorders caused by deficiencies of blood coagulation factors. The most common disorders are hemophilia A and B, resulting from deficiencies of blood coagulation factor VIII and IX, respectively. Another known bleeding disorder is von Willebrand's disease.

In plasma FVIII exists predominantly in a noncovalent complex with VWF and acts as a cofactor for activated factor IX in the membrane bound activated factor X generating complex.

Several attempts have been made to prolong the half-life of non-activated FVIII either by reducing its interaction with cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalently attaching polymers to FVIII (WO 94/15625, WO 97/11957 and U.S. Pat. No. 4,970,300), by encapsulation of FVIII (WO 99/55306), by introduction of novel metal binding sites (WO 97/03193), by covalently attaching the A2 domain to the A3 domain either by peptidic (WO 97/40145 and WO 03/087355) or disulfide linkage (WO 02/103024A2) or by covalently attaching the A1 domain to the A2 domain (WO2006/108590).

Another approach to enhance the functional half-life of FVIII or VWF is by PEGylation of FVIII (WO 2007/126808, WO 2006/053299, WO 2004/075923). PEGylation of VWF (WO 2006/071801) has also been attempted in an effort to indirectly enhance the half-life of FVIII present in plasma. Also fusion proteins of FVIII have been described (WO 2004/101740, WO2008/077616 and WO 2009/156137).

VWF, which is missing, functionally defective or only available in reduced quantity in different forms of von Willebrand disease (VWD), is a multimeric adhesive glycoprotein present in plasma, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The amino acid sequence and the cDNA sequence of wild-type VWF are disclosed in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741-residue pro-peptide and the 2050-residue polypeptide found in plasma (Fischer et al., FEBS Lett. 351: 345-348, 1994). After cleavage of the signal peptide in the endoplasmic reticulum a C-terminal disulfide bridge is formed between two monomers of VWF. During further transport through the secretory pathway 12 N-linked and 10 O-linked carbohydrate side chains are added. Importantly, VWF dimers are multimerized via N-terminal disulfide bridges and the propeptide of 741 amino acids is cleaved off by the enzyme PACE/furin in the late Golgi apparatus. The propeptide as well as the high-molecular-weight multimers of VWF (VWF-HMWM) are stored in the Weibel-Pallade bodies of endothelial cells or in the α-Granules of platelets.

Once secreted into plasma the protease ADAMTS13 cleaves VWF within the A1 domain of VWF. Plasma VWF consists of a range of multimers ranging from single dimers of 500 kDa to multimers consisting of more than 20 dimers of a molecular weight of over 10,000 kDa. Typically VWF high molecular weight multimers (VWF-HMWM) have the strongest hemostatic activity, which can be measured in ristocetin cofactor activity (VWF:RCo). The higher the ratio of VWF:RCo/VWF antigen, the higher the relative amount of high molecular weight multimers.

Defects in VWF are causal to von Willebrand disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely missing, VWD type 1 relates to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 relates to qualitative defects of VWF and can be as severe as VWD type 3. VWD type 2 has many sub forms some of them being associated with the loss or the decrease of high molecular weight multimers. Von VWD type 2a is characterized by a loss of both intermediate and large multimers. VWD type 2B is characterized by a loss of highest-molecular-weight multimers.

VWD is the most frequent inherited bleeding disorder in humans and can be treated by replacement therapy with concentrates containing VWF of plasma or recombinant origin. VWF can be prepared from human plasma as for example described in EP 05503991. EP 0784632 describes a method for producing and isolating recombinant VWF.

In plasma FVIII binds with high affinity to VWF, which protects it from premature catabolism and thus, plays in addition to its role in primary hemostasis, a crucial role in regulation of plasma levels of FVIII and as a consequence is also a central factor in the control of secondary hemostasis. The half-life of non-activated FVIII bound to VWF is about 12 to 14 hours in plasma. In von Willebrand disease type 3, where no or almost no VWF is present, the half-life of FVIII is only about 6 hours, leading to symptoms of mild to moderate hemophilia A in such patients due to decreased concentrations of FVIII. The stabilizing effect of VWF on FVIII has also been used to aid recombinant expression of FVIII in CHO cells (Kaufman et al. 1989, Mol Cell Biol).

SUMMARY OF THE INVENTION

In the current applicant's co-pending International Patent Application no. PCT/AU2015/050369 it is disclosed that a number of modifications in domain D′ of VWF can increase binding to Factor VIII. The disclosure of this application is included herein by cross-reference. The present inventors have now found that the binding of VWF to Factor VIII can be increased by other modifications in D′ and in particular by modifications in the D3 domain.

Accordingly in a first aspect the present invention provides a modified polypeptide which binds Factor VIII wherein the modified polypeptide comprises a sequence as shown in SEQ ID NO:3 or a fragment thereof in which the sequence comprises at least one modification in at least one position selected from the group consisting of L18, V42, K149, N248, S279, V320, T325, Q395 and K418; wherein the modified polypeptide binds to Factor VIII with an off rate lower than a reference polypeptide comprising an unmodified SEQ ID NO:3.

The present invention also provides a complex comprising a Factor VIII molecule and the modified polypeptide of the present invention and a polynucleotide encoding the modified polypeptide.

The present invention also provides a method of treating a bleeding disorder comprising administering to a patient in need thereof the modified polypeptide or complex of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Sample sensorgrams from the screen at neutral pH. The two candidates with strongest affinity and slowest off rate are circled.

FIG. 2: Sample sensorgrams showing detailed kinetics of CSL627 for two mutant D′D3-HSA candidates at pH7. a) Factor VIII binding D′D3-HSA with mutations: V1083A, S764G, S766Y; b) Factor VIII binding D′D3-HSA with mutations: S764G, S766Y; c) Factor VIII binding D′D3-HSA with mutations: V1083A, S764P, S766W; d) Factor VIII binding D′D3-HSA S764P, S766W.

FIG. 3: Sample sensorgrams showing detailed kinetics of Factor VIII for two mutant D′D3-HSA candidates at pH5.5 a) Factor VIII binding D′D3-HSA with mutations: V1083A, S764G, S766Y; b) Factor VIII binding D′D3-HSA with mutations: S764G, S766Y. c) Factor VIII binding D′D3-HSA with mutations: V1083A, S764P, S766W d) Factor VIII binding D′D3-HSA S764P, S766W.

FIG. 4: a) CSL627 binding D′D3-HSA Dimer with mutations: V1083A, S764E, S766Y at neutral pH ; b) CSL627 binding wildtype D′D3-HSA Dimer at neutral pH.

FIG. 5: a) CSL627 binding D′D3-HSA Dimer with mutations: V1083A, S764E, S766Y b) CSL627 binding wildtype D′D3-HSA Dimer at pH5.5.

DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

All publications mentioned in this specification are herein incorporated by reference in their entirety.

It must be noted that, as used in the subject specification, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a single agent, as well as two or more agents; reference to “a molecule” includes a single molecule, as well as two or more molecules; and so forth.

VWF

The term “von Willebrand Factor” or “VWF”, as used herein, refers to any polypeptide having a biological activity of wild type VWF, in particular the ability to bind Factor VIII. The gene encoding wild type VWF is transcribed into a 9 kb mRNA which is translated into a pre-propolypeptide of 2813 amino acids with an estimated molecular weight of 310,000 Da. The pre-propolypeptide contains a 22 amino acids signal peptide, a 741 amino acid pro-polypeptide and the mature subunit. Cleavage of the 741 amino acids propolypeptide from the N-terminus results in mature VWF consisting of 2050 amino acids. The amino acid sequence of the VWF pre-propolypeptide is shown in SEQ ID NO:2. Unless indicated otherwise, the amino acid numbering of VWF residues in this application refers to SEQ ID NO:2, even if the VWF molecule does not need to comprise all residues of SEQ ID NO:2. The amino acid sequence of mature VWF is shown in SEQ ID NO:4. The term “VWF” as used herein refers to the mature form of VWF unless indicated otherwise.

The propolypeptide of wild type VWF comprises multiple domains which are arranged in the following order:

-   -   D1-D2-D′-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK

The D1 and D2 domain represent the propeptide which is cleaved off to yield the mature VWF. The D′-D3 domains encompass amino acids responsible for binding to Factor VIII. The amino acid sequence of at least a portion of D′-D3 domains of wild type VWF is shown in SEQ ID NO:3. The carboxy terminal 90 residues comprise the “CK” domain that is homologous to the “cysteine knot” superfamily of protein. These family members have a tendency to dimerise through disulfide bonds.

Preferably, wild type VWF comprises the amino acid sequence of mature VWF as shown in SEQ ID NO:4. Also encompassed are additions, insertions, N-terminal, C-terminal or internal deletions of VWF as long as a biological activity of VWF, in particular the ability to bind FVIII, is retained. The biological activity is retained in the sense of the invention if the VWF with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type VWF. The biological activity of wild-type VWF can be determined by the artisan using methods for ristocetin co-factor activity (Federici A B et al. 2004. Haematologica 89:77-85), binding of VWF to GP Ibα of the platelet glycoprotein complex Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12:305-310), or a collagen binding assay (Kailas & Talpsep. 2001. Annals of Hematology 80:466-471). Where the biological activity of VWF is the ability to bind FVIII this can be measured in a number of ways, however, it is preferably measured as described in Example 1 herein.

Factor VIII

The terms “blood coagulation Factor VIII”, “Factor VIII” and “FVIII” are used interchangeably herein. “Blood coagulation Factor VIII” includes wild-type blood coagulation FVIII as well as derivatives of wild-type blood coagulation FVIII having the procoagulant activity of wild-type blood coagulation FVIII. Derivatives may have deletions, insertions and/or additions compared with the amino acid sequence of wild-type FVIII. The term FVIII includes proteolytically processed forms of FVIII, e.g. the form before activation, comprising heavy chain and light chain. Included are plasma derived and recombinant FVIII including B domain deleted FVIII. Examples of Commercial products include Advate®, Kogenate®, Xyntha®, Loctate® and Novoeight®.

The term “FVIII” includes any FVIII variants or mutants having at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type factor VIII.

As non-limiting examples, FVIII molecules include FVIII mutants preventing or reducing APC cleavage (Amano 1998. Thromb. Haemost. 79:557-563), FVIII mutants further stabilizing the A2 domain (WO 97/40145), FVIII mutants having increased expression (Swaroop et al. 1997. JBC 272:24121-24124), FVIII mutants having reduced immunogenicity (Lollar 1999. Thromb. Haemost. 82:505-508), FVIII reconstituted from differently expressed heavy and light chains (Oh et al. 1999. Exp. Mol. Med. 31:95-100), FVIII mutants having reduced binding to receptors leading to catabolism of FVIII like HSPG (heparan sulfate proteoglycans) and/or LRP (low density lipoprotein receptor related protein) (Ananyeva et al. 2001. TCM, 11:251-257), disulfide bond-stabilized FVIII variants (Gale et al., 2006. J. Thromb. Hemost. 4:1315-1322), FVIII mutants with improved secretion properties (Miao et al., 2004. Blood 103:3412-3419), FVIII mutants with increased cofactor specific activity (Wakabayashi et al., 2005. Biochemistry 44:10298-304), FVIII mutants with improved biosynthesis and secretion, reduced ER chaperone interaction, improved ER-Golgi transport, increased activation or resistance to inactivation and improved half-life (summarized by Pipe 2004. Sem. Thromb. Hemost. 30:227-237). Another particularly preferred example is a recombinant form of FVIII as described in Zollner et al 2013, Thrombosis Research, 132:280-287. All of these FVIII mutants and variants are incorporated herein by reference in their entirety.

Preferably FVIII comprises the full length sequence of FVIII as shown in SEQ ID NO:14. Also encompassed are additions, insertions, substitutions, N-terminal, C-terminal or internal deletions of FVIII as long as the biological activity of FVIII is retained. The biological activity is retained in the sense of the invention if the FVIII with modifications retains at least 10%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% of the biological activity of wild-type FVIII. The biological activity of FVIII can be determined by the artisan as described below.

A suitable test to determine the biological activity of FVIII is for example the one stage or the two stage coagulation assay (Rizza et al. 1982. Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophilias. NY Churchchill Livingston 1992) or the chromogenic substrate FVIII:C assay (S. Rosen, 1984. Scand J Haematol 33: 139-145, suppl.). The content of these references is incorporated herein by reference.

The amino acid sequence of the mature wild-type form of human blood coagulation FVIII is shown in SEQ ID NO:14. The reference to an amino acid position of a specific sequence means the position of said amino acid in the FVIII wild-type protein and does not exclude the presence of mutations, e.g. deletions, insertions and/or substitutions at other positions in the sequence referred to. For example, a mutation in “Glu2004” referring to SEQ ID NO:14 does not exclude that in the modified homologue one or more amino acids at positions 1 through 2332 of SEQ ID NO:14 are missing.

“FVIII” and/or “VWF” within the above definition also include natural allelic variations that may exist and occur from one individual to another. “FVIII” and/or “VWF” within the above definition further includes variants of FVIII and/or VWF. Such variants differ in one or more amino acid residues from the wild-type sequence. Examples of such differences may include conservative amino acid substitutions, i.e. substitutions within groups of amino acids with similar characteristics, e.g. (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids, and (6) aromatic amino acids. Examples of such conservative substitutions are shown in Table 1.

TABLE 1 (1) Alanine Glycine (2) Aspartic acid Glutamic acid (3) Asparagine Glutamine Serine Threonine (4) Arginine Histidine Lysine (5) Isoleucine Leucine Methionine Valine (6) Phenylalanine Tyrosine Tryptophan

Modified VWF

The modified VWF of the present invention has an amino acid sequence which differs from that of wild-type VWF. According to the present invention the modified VWF has at least one amino acid substitution within the D′-D3 domains, as compared to the amino acid sequence of the D′-D3 domains of wild-type VWF as shown in SEQ ID NO:3.

The amino acid sequence of the D′-D3 domains of the modified VWF can have one or more amino acid substitutions relative to SEQ ID NO:3. The amino acid sequence of the D′-D3 domains of the modified VWF preferably has one or 2 or 3 amino acid substitutions relative to SEQ ID NO:3.

It is preferred that the modified VWF includes a modification at position 320 of SEQ ID NO:3. It is also preferred that V at this position is substituted with A. In further preferred forms the modified VWF, in addition to modification at position 320, is also modified at position 1 and/or 3 of SEQ ID NO:3, preferably at both positions 1 and 3.

It is preferred that S at position 1 of SEQ ID NO:3 is substituted with an amino acid selected from the group consisting of G, P, V, E, Y, A and L.

It is also preferred that S at position 3 of SEQ ID NO:3 is substituted with an amino acid selected from the group consisting of Y, I, M, V, F, H, R and W.

Preferred combinations of substitutions include S764P/S766W/V1083A, S764G/S766Y/V1083A, S764E/S766Y/V1083A, N1011S/V1083A/K1181E, S766Y/V1083A, V805/Q1158L and K912E/T10885.

According to an aspect of this invention the binding affinity of the polypeptide of the present invention to FVIII is higher than that of a reference polypeptide which has the same amino acid sequence except for the modification in SEQ ID NO:3.

The binding affinity of a VWF molecule to a Factor VIII molecule can be determined by a binding assay used in the art. For example, the VWF molecule may be immobilized on a solid support, increasing concentrations of Factor VIII are applied, incubated for a certain period of time, and after washing, bound Factor VIII is determined with a chromogenic assay. The affinity constant or dissociation constant may then be determined by Scatchard analysis or another suitable method. A method of determining the affinity of binding of human Factor VIII to von Willebrand Factor are described in Vlot et al. (1995), Blood, Volume 85, Number 11, 3150-3157. Preferably, however, the affinity of VWF to Factor VIII is determined as described in Example 1 of this application.

Any indication herein of affinity, including dissociation constants, preferably refers to the binding of the modified VWF of the invention, or of the polypeptide of the invention to FVIII. The amino acid sequence of single chain of FVIII is shown in SEQ ID NO:14.

As the interaction of VWF with FVIII typically has a high on-rate, changes in the dissociation constant is largely dependent on changes in the off-rate. Accordingly the main focus in increasing the association of VWF with FVIII involves efforts to decrease the off-rate between FVIII and VWF. Preferably the off-rate of the modified VWF and FVIII in comparison to wild type VWF and FVIII is at least two fold lower, more preferably at least 5 fold lower, preferably at least 10 fold lower and more preferably at least 20 fold lower.

The dissociation constant of the complex consisting of VWF and FVIII is preferably 0.2 nmol/L or less, more preferably 0.175 nmol/L or less, more preferably 0.15 nmol/L or less, more preferably 0.125 nmol/L or less, more preferably 0.1 nmol/L or less, more preferably 0.05 nmol/L or less, most preferably 0.01 nmol/L or less.

The dissociation constant KD of a complex of the polypeptide of the invention and the Factor VIII of SEQ ID NO:14 is typically less than 90% of the dissociation constant KD of a complex of the reference polypeptide (e.g. the polypeptide of SEQ ID NO:4) and the Factor VIII of SEQ ID NO:14. The dissociation constant KD of a complex of the polypeptide of the invention and the Factor VIII of SEQ ID NO:14 is preferably less than 75%, more preferably less than 50%, more preferably less than 25%, more preferably less than 10%, more preferably less than 5%, of the dissociation constant KD of a complex of the reference polypeptide (e.g. the polypeptide of SEQ ID NO:4) and the Factor VIII of SEQ ID NO:14.

The reference polypeptide is a polypeptide the amino acid sequence of which is identical to that of the polypeptide of the present invention except for the mutation within the D′-D3 domains of VWF. That is, the reference polypeptide preferably has an amino acid sequence identical to that of the polypeptide of the present invention, with the proviso that the D′-D3 domains in the reference polypeptide consist of the amino acid sequence as shown in SEQ ID NO:3. In other words, the only difference in sequence between the polypeptide of the invention and the reference polypeptide lies in the amino acid sequence of the D′-D3 domains. The reference polypeptide has preferably been prepared under the same conditions as the polypeptide of the invention.

Half-Life Extending Moiety

The polypeptide of the present invention may consist of the modified VWF. In another embodiment, the polypeptide of the present invention may further comprise a half-life extending moiety. The half-life-extending moiety may be a heterologous amino acid sequence fused to the truncated VWF. Alternatively, the half-life-extending moiety may be chemically conjugated to the polypeptide comprising the truncated VWF by a covalent bond different from a peptide bond.

In certain embodiments of the invention, the half-life of the polypeptide of the invention is extended by chemical modification, e.g. attachment of a half-life extending moiety such as polyethylene glycol (PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids, elastin-like polypeptides, heparosan polymers or hyaluronic acid. In another embodiment, the polypeptide of the invention is conjugated to a HLEP such as albumin via a chemical linker. The principle of this conjugation technology has been described in an exemplary manner by Conjuchem LLC (see, e.g., U.S. Pat. No. 7,256,253).

Half-Life Enhancing Polypeptides (HLEPs)

Preferably, the half-life extending moiety is a half-life extending polypeptide (HLEP), more preferably HLEP is selected from albumin or fragments thereof, immunoglobulin constant region and portions thereof, e.g. the Fc fragment, solvated random chains with large hydrodynamic volume (e.g. XTEN (Schellenberger et al. 2009; Nature Biotechnol. 27:1186-1190), homo-amino acid repeats (HAP) or proline-alanine-serine repeats (PAS), afamin, alpha-fetoprotein, Vitamin D binding protein, transferrin or variants thereof, carboxyl-terminal peptide (CTP) of human chorionic gonadotropin-β subunit, polypeptides or lipids capable of binding under physiological conditions to albumin or immunoglobulin constant region.

A “half-life enhancing polypeptide” as used herein is preferably selected from the group consisting of albumin, a member of the albumin-family, the constant region of immunoglobulin G and fragments thereof, region and polypeptides capable of binding under physiological conditions to albumin, to members of the albumin family as well as to portions of an immunoglobulin constant region. It may be a full-length half-life-enhancing protein described herein (e.g. albumin, a member of the albumin-family or the constant region of immunoglobulin G) or one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or the biological activity of the coagulation factor. Such fragments may be of 10 or more amino acids in length or may include at least about 15, at least about 20, at least about 25, at least about 30, at least about 50, at least about 100, or more contiguous amino acids from the HLEP sequence or may include part or all of specific domains of the respective HLEP, as long as the HLEP fragment provides a functional half-life extension of at least 25% compared to the respective polypeptide without the HLEP.

The HLEP portion of the polypeptide of the invention may be a variant of a wild type HLEP. The term “variants” includes insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the FVIII-binding activity of the truncated VWF.

In particular, the proposed VWF HLEP fusion constructs of the invention may include naturally occurring polymorphic variants of HLEPs and fragments of HLEPs. The HLEP may be derived from any vertebrate, especially any mammal, for example human, monkey, cow, sheep, or pig. Non-mammalian HLEPs include, but are not limited to, hen and salmon.

In one embodiment the polypeptide has the following structure:

mVWF-L1-H,   [formula 1]

wherein mVWF is the modified VWF, L1 is a chemical bond or a linker sequence, and H is a HLEP.

L1 may be a chemical bond or a linker sequence consisting of one or more amino acids, e.g. of 1 to 50, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5 or 1 to 3 (e.g. 1, 2 or 3) amino acids and which may be equal or different from each other. Usually, the linker sequences are not present at the corresponding position in the wild-type VWF. Examples of suitable amino acids present in L1 include Gly and Ser. The linker should be non-immunogenic and may be a non-cleavable or cleavable linker. Non-cleavable linkers may be comprised of alternating glycine and serine residues as exemplified in WO2007/090584. In another embodiment of the invention the peptidic linker between the truncated VWF moiety and the albumin moiety consists of peptide sequences, which serve as natural interdomain linkers in human proteins. Preferably such peptide sequences in their natural environment are located close to the protein surface and are accessible to the immune system so that one can assume a natural tolerance against this sequence. Examples are given in WO2007/090584. Cleavable linker sequences are described, e.g., in WO 2013/120939 A1.

Preferred HLEP sequences are described infra. Likewise encompassed by the invention are fusions to the exact “N-terminal amino acid” or to the exact “C-terminal amino acid” of the respective HLEP, or fusions to the “N-terminal part” or “C-terminal part” of the respective HLEP, which includes N-terminal deletions of one or more amino acids of the HLEP. The polypeptide may comprise more than one HLEP sequence, e.g. two or three HLEP sequences. These multiple HLEP sequences may be fused to the C-terminal part of VWF in tandem, e.g. as successive repeats.

Albumin as HLEP

The terms, “human serum albumin” (HSA) and “human albumin” (HA) and “albumin” (ALB) are used interchangeably in this application. The terms “albumin” and “serum albumin” are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).

As used herein, “albumin” refers collectively to albumin polypeptide or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, “albumin” refers to human albumin or fragments thereof, especially the mature form of human albumin as shown in SEQ ID NO:16 herein or albumin from other vertebrates or fragments thereof, or analogs or variants of these molecules or fragments thereof.

In particular, the proposed polypeptides of the invention may include naturally and non-naturally occurring polymorphic variants of human albumin and fragments of human albumin. Generally speaking, an albumin fragment or variant will be at least 10, preferably at least 40, most preferably more than 70 amino acids long.

Preferred embodiments of the invention include albumin variants used as a HLEP of the polypeptide of the invention with enhanced binding to the FcRn receptor. Such albumin variants may lead to a longer plasma half-life of a truncated VWF albumin variant fusion protein as compared to a truncated VWF fusion with a wild-type albumin. Variants include those described in WO 2014072481, WO 2012150319, WO 2013135896, WO 2011124718, WO 2011051489 and WO 2012059486, the disclosures of which are incorporated by cross-reference.

The albumin portion of the polypeptides of the invention may comprise at least one subdomain or domain of HA or conservative modifications thereof.

Immunoglobulins as HLEPs

Immunoglobulin G (IgG) constant regions (Fc) are known in the art to increase the half-life of therapeutic proteins (Dumont J A et al. 2006. BioDrugs 20:151-160). The IgG constant region of the heavy chain consists of 3 domains (CH1-CH3) and a hinge region. The immunoglobulin sequence may be derived from any mammal, or from subclasses IgG1, IgG2, IgG3 or IgG4, respectively. IgG and IgG fragments without an antigen-binding domain may also be used as HLEPs. The therapeutic polypeptide portion is connected to the IgG or the IgG fragments preferably via the hinge region of the antibody or a peptidic linker, which may even be cleavable. Several patents and patent applications describe the fusion of therapeutic proteins to immunoglobulin constant regions to enhance the therapeutic protein's in vivo half-lives. US 2004/0087778 and WO 2005/001025 describe fusion proteins of Fc domains or at least portions of immunoglobulin constant regions with biologically active peptides that increase the half-life of the peptide, which otherwise would be quickly eliminated in vivo. Fc-IFN-β fusion proteins were described that achieved enhanced biological activity, prolonged circulating half-life and greater solubility (WO 2006/000448). Fc-EPO proteins with a prolonged serum half-life and increased in vivo potency were disclosed (WO 2005/063808) as well as Fc fusions with G-CSF (WO 2003/076567), glucagon-like peptide-1 (WO 2005/000892), clotting factors (WO 2004/101740) and interleukin-10 (U.S. Pat. No. 6,403,077), all with half-life enhancing properties.

Various HLEPs which can be used in accordance with this invention are described in detail in WO 2013/120939 A1, the disclosure of which is included herein by cross-reference.

Linker Sequences

According to this invention, the therapeutic polypeptide moiety may be coupled to the HLEP moiety by a peptide linker. The linker should be non-immunogenic and may be a non-cleavable or cleavable linker.

Non-cleavable linkers may be comprised of alternating glycine and serine residues as exemplified in WO2007/090584.

In another embodiment of the invention the peptidic linker between the VWF moiety and the albumin moiety consists of peptide sequences, which serve as natural interdomain linkers in human proteins. Preferably such peptide sequences in their natural environment are located close to the protein surface and are accessible to the immune system so that one can assume a natural tolerance against this sequence. Examples are given in WO2007/090584.

Cleavable linkers should be flexible enough to allow cleavage by proteases. In a preferred embodiment the cleavage of the linker proceeds comparably fast as the activation of FVIII within the fusion protein, if the fusion protein is a modified FVIII.

The cleavable linker preferably comprises a sequence derived from

(a) the therapeutic polypeptide to be administered itself if it contains proteolytic cleavage sites that are proteolytically cleaved during activation of the therapeutic polypeptide,

(b) a substrate polypeptide cleaved by a protease which is activated or formed by the involvement of the therapeutic polypeptide, or

(c) a polypeptide involved in coagulation or fibrinolysis.

The linker region in a more preferred embodiment comprises a sequence of VWF, which should result in a decreased risk of neoantigenic properties of the expressed fusion protein.

The linker peptides are preferably cleavable by the proteases of the coagulation system, for example FIIa, FIXa, FXa, FXIa, FXIIa and FVIIa.

Exemplary combinations of therapeutic polypeptide, cleavable linker and HLEP include the constructs listed in WO2007/090584 (for example in table 2 and FIG. 4) and WO2007/144173 (for example in table 3a and 3b), but are not limited to these.

In another embodiment, the functional half-life of polypeptide of the invention or of FVIII complexed with the polypeptide of the invention is prolonged compared to that of wild type VWF or to that of FVIII complexed with wild type VWF, or with the reference polypeptide as defined supra. The increase may be more than 15%, for example at least 20% or at least 50%. Again, such functional half-life values can be measured in vitro in blood samples taken at different time intervals from said mammal after the modified VWF or the complex of FVIII with modified VWF has been administered.

In another embodiment of the invention, the polypeptide of the invention or FVIII complexed with the polypeptide of the invention exhibits an improved in vivo recovery compared to wild type VWF or to FVIII complexed with wild type VWF, or with the reference polypeptide defined supra. The in vivo recovery can be determined in vivo for example in normal animals or in animal models of hemophilia A, like FVIII knockout mice in which one would expect an increased percentage of FVIII be found by antigen or activity assays in the circulation shortly (5 to 10 min.) after i.v. administration compared to the corresponding wild-type VWF, or reference polypeptide defined supra.

The in vivo recovery is preferably increased by at least 10%, more preferably by at least 20%, and even more preferably by at least 40% compared to FVIII complexed with wild-type VWF, or with the reference polypeptide defined supra.

Polynucleotides

The invention further relates to a polynucleotide encoding a modified VWF or a polypeptide comprising said modified VWF, as described in this application. The term “polynucleotide(s)” generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. The polynucleotide may be single- or double-stranded DNA, single or double-stranded RNA. As used herein, the term “polynucleotide(s)” also includes DNAs or RNAs that comprise one or more modified bases and/or unusual bases, such as inosine. It will be appreciated that a variety of modifications may be made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term “polynucleotide(s)” as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.

The skilled person will understand that, due to the degeneracy of the genetic code, a given polypeptide can be encoded by different polynucleotides. These “variants” are encompassed by this invention.

Preferably, the polynucleotide of the invention is an isolated polynucleotide. The term “isolated” polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides may be purified from a host cell. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

The invention further relates to a group of polynucleotides which together encode the modified VWF of the invention, or the polypeptide of the invention comprising the modified VWF. A first polynucleotide in the group may encode the N-terminal part of the modified VWF, and a second polynucleotide may encode the C-terminal part of the modified VWF.

Yet another aspect of the invention is a plasmid or vector comprising a polynucleotide according to the invention. Preferably, the plasmid or vector is an expression vector. In a particular embodiment, the vector is a transfer vector for use in human gene therapy.

The invention also relates to a group of plasmids or vectors that comprise the above group of polynucleotides. A first plasmid or vector may contain said first polynucleotide, and a second plasmid or vector may contain said second polynucleotide. Alternatively, both coding sequences are cloned into one expression vector either using two separate promoter sequences or one promoter and an internal ribosome entry site (IRES) element which may be used for example to direct the expression of furin to enhance the generation of mature VWF.

Still another aspect of the invention is a host cell comprising a polynucleotide, a plasmid or vector of the invention, or a group of polynucleotides or a group of plasmids or vectors as described herein.

The host cells of the invention may be employed in a method of producing a modified VWF or a polypeptide comprising said modified VWF, which is part of this invention. The method comprises:

-   -   (a) culturing host cells of the invention under conditions such         that the desired modified protein is expressed; and     -   (b) optionally recovering the desired modified protein from the         host cells or from the culture medium.

It is preferred to purify the modified VWF of the present invention, or the polypeptide comprising the modified VWF to ≥80% purity, more preferably ≥95% purity, and particularly preferred is a pharmaceutically pure state that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, an isolated or purified modified VWF of the invention or polypeptide of the invention is substantially free of other, non-related polypeptides.

The various products of the invention are useful as medicaments. Accordingly, the invention relates to a pharmaceutical composition comprising a modified VWF or a polypeptide comprising said modified VWF as described herein, a polynucleotide of the invention, or a plasmid or vector of the invention.

The invention also concerns a method of treating an individual suffering from a blood coagulation disorder such as hemophilia A or B or VWD. The method comprises administering to said individual an efficient amount of (i) FVIII and of the modified VWF or the polypeptide comprising the modified VWF or (ii) of the complex of FVIII with modified VWF or (iii) of the complex of FVIII with the polypeptide comprising modified VWF as described herein. In another embodiment, the method comprises administering to the individual an efficient amount of a polynucleotide of the invention or of a plasmid or vector of the invention. Alternatively, the method may comprise administering to the individual an efficient amount of the host cells of the invention described herein.

Expression of the Modified Polypeptides

The production of recombinant mutant proteins at high levels in suitable host cells requires the assembly of the above-mentioned modified polynucleotides, typically cDNA, into efficient transcriptional units together with suitable regulatory elements in a recombinant expression vector that can be propagated in various expression systems according to methods known to those skilled in the art. Efficient transcriptional regulatory elements could be derived from viruses having animal cells as their natural hosts or from the chromosomal DNA of animal cells. Preferably, promoter-enhancer combinations derived from the Simian Virus 40, adenovirus, BK polyoma virus, human cytomegalovirus, or the long terminal repeat of Rous sarcoma virus, or promoter-enhancer combinations including strongly constitutively transcribed genes in animal cells like beta-actin or GRP78 can be used. In order to achieve stable high levels of mRNA transcribed from the cDNAs, the transcriptional unit should contain in its 3′-proximal part a DNA region encoding a transcriptional termination-polyadenylation sequence. Preferably, this sequence is derived from the Simian Virus 40 early transcriptional region, the rabbit beta-globin gene, or the human tissue plasminogen activator gene.

The cDNAs are then integrated into the genome of a suitable host cell line for expression of the modified FVIII and/or VWF proteins. Preferably this cell line should be an animal cell-line of vertebrate origin in order to ensure correct folding, disulfide bond formation, asparagine-linked glycosylation and other post-translational modifications as well as secretion into the cultivation medium. Examples on other post-translational modifications are tyrosine O-sulfation and proteolytic processing of the nascent polypeptide chain. Examples of cell lines that can be used are monkey COS-cells, mouse L-cells, mouse C127-cells, hamster BHK-21 cells, human embryonic kidney 293 cells, and hamster CHO-cells.

The recombinant expression vector encoding the corresponding cDNAs can be introduced into an animal cell line in several different ways. For instance, recombinant expression vectors can be created from vectors based on different animal viruses. Examples of these are vectors based on baculovirus, vaccinia virus, adenovirus, and preferably bovine papilloma virus.

The transcription units encoding the corresponding DNA's can also be introduced into animal cells together with another recombinant gene which may function as a dominant selectable marker in these cells in order to facilitate the isolation of specific cell clones which have integrated the recombinant DNA into their genome. Examples of this type of dominant selectable marker genes are Tn5 amino glycoside phosphotransferase, conferring resistance to gentamycin (G418), hygromycin phosphotransferase, conferring resistance to hygromycin, and puromycin acetyl transferase, conferring resistance to puromycin. The recombinant expression vector encoding such a selectable marker can reside either on the same vector as the one encoding the cDNA of the desired protein, or it can be encoded on a separate vector which is simultaneously introduced and integrated to the genome of the host cell, frequently resulting in a tight physical linkage between the different transcription units.

Other types of selectable marker genes which can be used together with the cDNA of the desired protein are based on various transcription units encoding dihydrofolate reductase (dhfr). After introduction of this type of gene into cells lacking endogenous dhfr-activity, preferentially CHO-cells (DUKX-B11, DG-44), it will enable these to grow in media lacking nucleosides. An example of such a medium is Ham's F12 without hypoxanthine, thymidine, and glycine. These dhfr-genes can be introduced together with the FVIII cDNA transcriptional units into CHO-cells of the above type, either linked on the same vector or on different vectors, thus creating dhfr-positive cell lines producing recombinant protein.

If the above cell lines are grown in the presence of the cytotoxic dhfr-inhibitor methotrexate, new cell lines resistant to methotrexate will emerge. These cell lines may produce recombinant protein at an increased rate due to the amplified number of linked dhfr and the desired protein's transcriptional units. When propagating these cell lines in increasing concentrations of methotrexate (1-10000 nM), new cell lines can be obtained which produce the desired protein at very high rate.

The above cell lines producing the desired protein can be grown on a large scale, either in suspension culture or on various solid supports. Examples of these supports are micro carriers based on dextran or collagen matrices, or solid supports in the form of hollow fibres or various ceramic materials. When grown in cell suspension culture or on micro carriers the culture of the above cell lines can be performed either as a bath culture or as a perfusion culture with continuous production of conditioned medium over extended periods of time. Thus, according to the present invention, the above cell lines are well suited for the development of an industrial process for the production of the desired recombinant mutant proteins

Purification and Formulation

The recombinant modified VWF protein, which accumulates in the medium of secreting cells of the above types, can be concentrated and purified by a variety of biochemical and chromatographic methods, including methods utilizing differences in size, charge, hydrophobicity, solubility, specific affinity, etc. between the desired protein and other substances in the cell cultivation medium.

An example of such purification is the adsorption of the recombinant mutant protein to a monoclonal antibody, directed to e.g. a HLEP, preferably human albumin, or directed to the respective coagulation factor, which is immobilised on a solid support. After adsorption of the modified VWF to the support, washing and desorption, the protein can be further purified by a variety of chromatographic techniques based on the above properties.

The order of the purification steps is chosen e.g. according to capacity and selectivity of the steps, stability of the support or other aspects. Preferred purification steps include but are not limited to ion exchange chromatography steps, immune affinity chromatography steps, affinity chromatography steps, hydrophobic interaction chromatography steps, dye chromatography steps, hydroxyapatite chromatography steps, multimodal chromatography steps, and size exclusion chromatography steps.

In order to minimize the theoretical risk of virus contaminations, additional steps may be included in the process that provide effective inactivation or elimination of viruses. Such steps e.g. are heat treatment in the liquid or solid state, treatment with solvents and/or detergents, radiation in the visible or UV spectrum, gamma-radiation or nanofiltration.

The modified polynucleotides (e.g. DNA) of this invention may also be integrated into a transfer vector for use in the human gene therapy.

The various embodiments described herein may be combined with each other. The present invention will be further described in more detail in the following examples thereof. This description of specific embodiments of the invention will be made in conjunction with the appended figures.

The modified VWF as described in this invention can be formulated into pharmaceutical preparations for therapeutic use. The purified protein may be dissolved in conventional physiologically compatible aqueous buffer solutions to which there may be added, optionally, pharmaceutical excipients to provide pharmaceutical preparations.

Such pharmaceutical carriers and excipients as well as suitable pharmaceutical formulations are well known in the art (see for example “Pharmaceutical Formulation Development of Peptides and Proteins”, Frokjaer et al., Taylor & Francis (2000) or “Handbook of Pharmaceutical Excipients”, 3rd edition, Kibbe et al., Pharmaceutical Press (2000)). Standard pharmaceutical formulation techniques are well known to persons skilled in the art (see, e.g., 2005 Physicians' Desk Reference®, Thomson Healthcare: Montvale, N.J., 2004; Remington: The Science and Practice of Pharmacy, 20th ed., Gennaro et al., Eds. Lippincott Williams & Wilkins: Philadelphia, Pa., 2000). In particular, the pharmaceutical composition comprising the polypeptide variant of the invention may be formulated in lyophilized or stable liquid form. The polypeptide variant may be lyophilized by a variety of procedures known in the art. Lyophilized formulations are reconstituted prior to use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution.

Formulations of the composition are delivered to the individual by any pharmaceutically suitable means of administration. Various delivery systems are known and can be used to administer the composition by any convenient route. Preferentially, the compositions of the invention are administered systemically. For systemic use, the proteins of the invention are formulated for parenteral (e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonary, intranasal or transdermal) or enteral (e.g., oral, vaginal or rectal) delivery according to conventional methods. The most preferential routes of administration are intravenous and subcutaneous administration. The formulations can be administered continuously by infusion or by bolus injection. Some formulations encompass slow release systems.

The proteins of the present invention are administered to patients in a therapeutically effective dose, meaning a dose that is sufficient to produce the desired effects, preventing or lessening the severity or spread of the condition or indication being treated without reaching a dose which produces intolerable adverse side effects. The exact dose depends on many factors as e.g. the indication, formulation, and mode of administration and has to be determined in preclinical and clinical trials for each respective indication.

The pharmaceutical composition of the invention may be administered alone or in conjunction with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical. One example of such an agent is the combination of modified VWF with FVIII.

A summary of the sequences referred to herein is set out in Table 2.

TABLE 2 SEQ ID NO: Description 1 Nucleotide sequence of DNA encoding SEQ ID NO: 2 2 Amino acid sequence of human VWF pre-propolypeptide 3 Amino acid sequence of D′-D3 domains of human VWF 4 Amino acid sequence of mature human VWF 5 D′-D3 including mutations S764P/S766W/V1083A 6 D′-D3 including mutations S764G/S766Y/V1083A 7 D′-D3 including mutations S764E/S766Y/V1083A 8 D′-D3 including mutations N1011S/V1083A/K1181E 9 D′-D3 including mutation V1083A 10 D′-D3 including mutation S1042T 11 D′-D3 including mutations V805A/Q1158L 12 D′-D3 including mutations K912E/T1088S 13 D′-D3 including mutation L781P 14 Amino acid sequence of human Factor VIII 15 Amino acid sequence of a mature single-chain Factor VIII 16 Amino acid sequence of human serum albumin 17 D′-D3 including mutations S766Y/V1083A

EXAMPLES Example 1

vWF Mutants with Improved FVIII Binding

Background

As discussed above and in co-pending International Patent Application No. PCT/AU2015/050369 the majority of circulating FVIII is in complex with VWF. In humans, FVIII is cleared from the blood with a t½ of approximately 2 hr and 16 hr in the absence and presence of VWF, respectively. Although VWF imparts an increase in FVIII half-life, it also places an upper limit on the t½ that is dictated by its own half-life. U.S. Pat. No. 8,575,104 discloses a VWF-albumin fusion protein. This fusion protein has a five-fold longer half-life than wild type VWF in a rodent model. A stable complex between this fusion protein and FVIII may confer additional half-life benefits for FVIII. Although the equilibrium binding constant for the FVIII/vWF interaction is high, the binding kinetics are rapid and any FVIII in complex with the VWF-albumin fusion protein will quickly exchange with endogenous vWF upon infusion. Accordingly if the off-rate of FVIII with VWF-albumin fusion is substantially equivalent to the off-rate of FVIII with native VWF then the use of the VWF-albumin fusion will not provide any substantial increase in the half life of FVIII.

Accordingly, in order to take advantage of the longer half life of the VWF-albumin fusion to extend the half life of FVIII it is necessary to decrease the off-rate of FVIII with the VWF-albumin fusion. From modeling studies taking advantage of measurement made in patients with Type 2N von Willebrand disease in which the level of VWF is normal but the ability of the VWF to associate with FVIII is severely diminished it has been estimated that at least a five fold decrease in off-rate is required to provide a clinically relevant improvement in FVIII half life. The postulated relationship between decrease in FVIII VWF-albumin fusion off-rate and increase in FVIII half life is set out in Table 3.

TABLE 3 Postulated increase in FVIII half life (For 50 IU/kg of FVIII and Decrease in FVIII VWF-albumin 100 IU/kg of VWF with the fusion off-rate VWF 5x half life extended) 2 fold 2.2 3 fold 2.6 5 fold 3 10 fold  3.6 20 fold  4.1

In an effort to decrease FVIII VWF-albumin fusion off-rate experiments were conducted to assess whether mutant VWF-albumin fusion protein may provide a significantly slower FVIII off-rate thereby providing a viable option to extend the half-life of FVIII through stable association with the VWF-albumin fusion protein.

In an extension of the work described in PCT/AU2015/050369 further mutations and combinations of mutations were investigated with an emphasis on modifications in the D3 domain. In these experiments a recombinant form of FVIII was used. This FVIII is described in Zollner et al 2013, Thrombosis Research, 132:280-287.

Methods

A synthetic, codon-optimised cDNA encoding the D′ and D3 domains of human von Willebrand Factor (vWF; amino acids (aa) 764-1242 (SEQ ID NO:2); based on GenBank accession no. NP 000543) was obtained from GeneART AG (Regensberg, Germany). This was modified at the 5′ end to encode its own signal peptide (aa1-22) and at the 3′ end to encode a human serum albumin (HAS) via a glycine serine linker. The construct (Hu-vWF[764-1270]-HSA) was directionally cloned into the pcDNA3.1 mammalian expression vector (Invitrogen, USA) with a Kozak consensus sequence (GCCACC) upstream of the initiating methionine and a double stop codon (TGA) at the 3′ end of the open reading frame, and the plasmid sequence confirmed by automated sequencing. This expression plasmid was then used as a template to make single, double or triple residue changes using standard PCR techniques and the constructs cloned into pcDNA3.1 and sequenced as described above. A second codon-optimised cDNA encoding the D1 and D2 domains (aa1-762) of Hu-vWF with a C-terminal FLAG tag (DYKDDDDK) was also synthesized and obtained from GeneArt; this was cloned as above into pcDNA3.1 and sequenced.

For transient mammalian expression, Freestyle™ 293 suspension cells (Invitrogen] were grown to 1.1×10⁶ cells/ml in 5 ml Freestyle Expression media (Invitrogen). 7 μL 293Fectin (Invitrogen) transfection reagent was pre-incubated for 5 minutes with 167 μL Opti-MEM I medium (Invitrogen), then added to 2.5 μg plasmid DNA encoding wild-type/mutant Hu-vWF[764-1242]-HSA plus 2.5 μg plasmid DNA encoding Hu-vWF[1-762]-FLAG and the mixture incubated for a further 20 minutes. The DNA-293Fectin complex was added to the cells which were cultured for 6 days at 37° C., 8% CO₂ in a shaking incubator at 250 rpm. Culture supernatants were harvested by centrifugation at 2000 rpm for 5 minutes and stored at 4° C. for analysis.

Mouse anti-HSA antibody was immobilized on a CMS chip using standard NHS/EDC coupling chemistry. Typically, the immobilization level was about 14,000 RU. Each mutant of vWF-HSA was captured on a single spot in each flow cell for 2 minutes at various concentrations ranging from 0.1-1 μg/ml. Capture levels ranged from 100-200 RU. An adjacent spot in which anti-HSA was immobilized, but no vWF-HSA captured was used as a reference. Capture was performed every cycle, before FVIII binding analysis.

An initial experiment was set up at neutral pH to screen a series of mutations for the top affinity and off rate improved vWF-HSA mutants. Factor VIII was injected at random and in duplicate over all spots and all flow cells in use at 5, 1 and 1.25 nM. The results of this screen are set out in Table 4.

TABLE 4 Screen at pH 7: affinities and kinetic rates of Factor VIII for various mutant D′D3-HSA dimer proteins ranked from strongest to weakest affinities. Sample Protein ka (1/Ms) kd (1/s) KD (M) size V1083A, S764P, S766W 6.15E+06 1.17E−04 1.90E−11 n = 1 V1083A, S764G, S766Y 6.19E+06 1.86E−04 3.01E−11 n = 2 N1011S, V1083A, K1181E 5.31E+06 4.30E−04 8.10E−11 n = 1 V1083A 5.51E+06 5.39E−04 9.78E−11 n = 1 S1042T 4.39E+06 4.69E−04 1.07E−10 n = 2 V805A, Q1158L 4.26E+06 6.32E−04 1.49E−10 n = 2 K912E, T1088S 5.17E+06 8.00E−04 1.55E−10 n = 2 L781P 4.27E+06 6.99E−04 1.64E−10 n = 2 WT 4.83E+06 1.08E−03 2.23E−10 n = 1 R960G 4.09E+06 1.11E−03 2.72E−10 n = 2 WT = wildtype

EXAMPLE 2 Detailed Kinetic Analysis

Subsequent experiments were conducted using purified material where detailed kinetic analysis at pH7 was set up for the top two candidates, including controls. vWF-HAS proteins were purified using Capture Select™ Human Albumin affinity resin and then the vWF-HAS dimer further purified by preparation Size Exclusion Chromatography. Purified material was then stored at 4° C. prior to analysis. Capture of the vWF-HAS dimer was performed as before and Factor VIII then injected at 1, 0.5, 0.25, 0.125 and 0.06 nM. In a similar manner detailed kinetic analysis on the top two candidates, including controls was set up at pH5.5 where Factor VIII was injected at various concentrations that best suited the interaction.

Throughout all experiments buffer blanks were also injected over all captured proteins. The association and dissociation of Factor VIII was monitored for 3 minutes respectively during the “screening” experiment. The association of CSL627 was monitored for 5 minutes and dissociation was monitored for 20 and 60 minutes during the “detailed kinetic analysis” experiments at neutral pH. At pH 5.5 the association and dissociation of Factor VIII was monitored for various time frames that best suited the interaction.

Post the dissociation period the surface was regenerated with a 45 second injection of 25 mM Glycine pH2.6. Running buffer throughout was 10 mM HEPES, 150 mM NaCl, 10 mM Na Citrate, 2.5 mM CaCl₂, 0.1% BSA, pH7.3 and pH5.5, while the flow rate was 30 μl/min. Each interaction was measured at least 2 times (n=2) at 37° C.

Responses for binding to the reference spot were subtracted from those of the vWF-HSA captured spots. Responses from blank injections were then subtracted from those of all other samples to produce double-referenced sensorgrams. Double referenced sensorgrams were fitted to a 1:1 kinetic model, including a term for mass transport limitation. Association and dissociation rates were fitted globally and Rmax fitted locally. The results are set out in Tables 5 and 6 and shown in FIGS. 1-3.

TABLE 5 Detailed Kinetics at pH 7: affinities and kinetic rates of Factor VIII for mutant D′D3-HSA dimers. Protein ka (1/Ms) kd (1/s) KD (M) Sample Size V1083A, S764P, 1.15E+07 3.84E−05 3.36E−12 n = 4 S766W (PWA) V1083A, S764G, 1.63E+07 7.30E−05 4.50E−12 n = 2 S766Y (GYA) S764G, S766Y (GY) 2.95E+07 3.65E−04 1.25E−11 n = 3 WT* 1.06E+07 8.46E−04 8.03E−11 n = 2 WT = wildtype

TABLE 6 Detailed Kinetics at pH5.5: affinities and kinetic rates of Factor VIII for mutant D′D3-HSA dimers Sample Protein ka (1/Ms) kd (1/s) KD (M) Size V1083A, S764P, S766W 2.51E+06 8.44E−04 3.42E−10 n = 4 S764P, S766W 2.94E+06 1.80E−03 6.14E−10 n = 2 V1083A, S764G, S766Y 2.05E+06 8.12E−03 4.05E−09 n = 4 S764G, S766Y 2.57E+06 2.50E−02 1.13E−08 n = 3

Example 3 Further Kinetic Analysis Method in Brief:

Subsequent experiments were conducted where further detailed kinetic analysis at pH 7 and pH 5.5 was set up for the top candidate proteins produced using a single DNS construct encoding the D1D2D′D3 region of native vWF fused to HAS (Hu-vWF [1-1242]-HAS). As before, this construct was used as a template to make single, double or triple residue changes. Transfection and purification of vWF-HAS dimers was performed as before.

Mouse anti-HSA antibody was immobilized on a CMS chip using standard NHS/EDC coupling chemistry. Typically, the immobilization level was about 14,000 RU. Each Dimer mutant of D′D3-HSA was captured on a single spot in each flow cell for 2 minutes at various concentrations ranging from 0.2-0.7 μg/ml. Capture levels ranged from 50-250 RU. An adjacent spot in which anti-HSA was immobilized, but no D′D3-HSA captured was used as a reference. Capture was performed every cycle, before FVIII (CSL627) binding analysis.

CSL627 was injected at random and in duplicate over all spots in all flow cells. At neutral pH CSL627 was injected at 1, 0.5, 0.25, 0.125 and 0.06 nM. The association was monitored for 5 minutes, while the dissociation was monitored for 20 minutes as well as for 1 hour at the 1 nM concentration. Buffer blanks were also injected. At pH5.5 CSL627 was injected at various concentrations and time frames that best suited the interaction taking place.

After the dissociation period the surface was regenerated with a 45 second injection of 25 mM Glycine pH2.6. Running buffer throughout was 10 mM HEPES, 150 mM NaCl, 10 mM Na Citrate, 2.5 mM CaCl2, 0.1% BSA, pH7.3 and pH5.5, while the flow rate was 30 μl/min. Each interaction was measured 4 times (n=4) at 37° C.

Responses for binding to the reference spot were subtracted from those of the vWF-HSA captured spots. Responses from blank injections were then subtracted from those of all other samples to produce double-referenced sensorgrams. Double referenced sensorgrams were fitted to a 1:1 kinetic model, including a term for mass transport limitation. Association and dissociation rates were fitted globally and Rmax fitted locally.

The results are set out in Tables 7 and 8 and FIGS. 4 and 5.

TABLE 7 Detailed Kinetics at pH 7: affinities and kinetic rates of CSL627 (Factor VIII) binding to mutant D′D3-HSA dimers. vWF D′D3-HSA Dimer ka (1/Ms) kd (1/s) KD (M) Sample Size (n) S764G, S766Y, V1083A 1.33E+07 ± 1.58E+06 6.44E−05 ± 3.39E−06 4.96E−12 ± 3.31E−13 n = 4 S764E, S766Y, V1083A 8.59E+06 ± 4.21E+05 4.77E−05 ± 3.59E−06 5.65E−12 ± 5.82E−13 n = 5 S766Y, V1083A 1.47E+07 ± 1.2E+06 8.88E−05 ± 7.15E−06 6.05E−12 ± 2.53E−13 n = 4 S764E, V1083A 1.77E+07 ± 1.74E+06 2.07E−04 ± 3.04E−05 1.16E−11 ± 1.03E−12 n = 4 S764G, V1083A 1.89E+07 ± 7.01E+05 3.59E−04 ± 8.59E−06 1.90E−11 ± 3.74E−13 n = 4 Wildtype 2.31E+07 ± 3.03E−06 2.22E−03 ± 1.02E−04 9.88E−11 ± 9.8E−12  n = 4

TABLE 8 Detailed Kinetics at pH5.5: affinities and kinetic rates of CSL627 binding to mutant D′D3-HSA dimers. vWF D′D3-HSA Dimer ka (1/Ms) kd (1/s) KD (M) Sample Size (n) S764E, S766Y, V1083A 3.61E+06 ± 2.12E+05 1.89E−03 ± 6.42E−05 5.35E−10 ± 2.64E−11 n = 4 S766Y, V1083A 4.04E+06 ± 4.64E+05 9.55E−03 ± 7.47E−04 2.38E−09 ± 8.42E−11 n = 3 S764G, S766Y, V1083A 6.81E+06 ± 3.77E+06 1.56E−02 ± 6.27E−03 2.93E−09 ± 4.70E−10 n = 4 S764E, V1083A 2.50E+06 ± 1.67E+05 2.07E−02 ± 7.8E−04  8.32E−09 ± 3.12E−10 n = 4 S764G, V1083A 9.08E+05 ± 3.54E+04 7.44E−02 ± 1.37E−03 8.24E−08 ± 4.18E−09 n = 4 Wildtype 6.48E+05 ± 2.32E+04 7.91E−02 ± 9.81E−03 1.22E−07 ± 1.36E−08 n = 3

Example 4 PK Analysis and Impact on FVIII Half-Life Methods

A stable CHO derived cell line expressing the Hu vWF D′D3-FP S764E; S766Y variant was generated using standard experimental methods. Material was produced from the stable cell line in a 10 L bioreactor and vWF D′D3-FP S764E; S766Y dimer purified as previously described.

To assess the relative impact of wild-type and the vWF D′D3-FP S764E; S766Y variant on FVIII levels, CD Rats (3 animals/group) were given a combination of recombinant FVIII (CSL627 at 200 IU/kg) and vWF-FP proteins at the doses shown in Table 9. Plasma samples were taken at 0, 3, 8, 24, 48, 56 and 72 hours following iv administration and FVIII levels determined using an Asserachrom FVIII:Ag ELISA. This data was then used to determine the FVIII Half-life and Mean Residence Times given in Table 9.

TABLE 9 PK Analysis: FVIII Half-life and Mean Residence time following co administration of recombinant FVIII and D′D3-HSA dimers Mean Residence Treatment group Time T½ (readout based on Asserachrom FVIII:Ag ELISA) (hrs) (hrs) CSL627-rVIII-SingleChain, 200 IU/kg 10.3 7.1 rD′D3-FP S764E; S766Y 0.09 mg/kg + CSL627 13.1 9.1 200 IU/kg rD′D3-FP S764E; S766Y 0.3 mg/kg + CSL627 17.8 12.3 200 IU/kg rD′D3-FP S764E; S766Y 0.9 mg/kg + CSL627 22.6 15.6 200 IU/kg rD′D3-FP wild type 1.0 mg/kg + CSL627 200 IU/kg 14.1 10.1 rD′D3-FP wild type 3.0 mg/kg + CSL627 200 IU/kg 18.4 12.7 rD′D3-FP wild type 10.0 mg/kg + CSL627 200 IU/kg 26.2 18.1

CONCLUSION

From the initial screen D′D3-HSA with mutations: S764P, S766W, V1083A (referred as PWA mutant) and S764G, S766Y, V1083A (referred to as GYA mutant) appeared to have the strongest affinity and slowest off rate for Factor VIII.

At neutral pH, vWF D′D3-HSA mutant dimers with the most improved affinity and off rate for CSL627 (Factor VIII) are S764G/S766Y/V1083A (GYA), S764E/S766Y/V1083A (EYA) and S766Y/V1083A (YA) with a 5 pM KD and a 10⁻⁵ l/s off rate. This is about a 20 fold improvement in affinity and 40 fold improvement in off rate compared to the wild-type dimer.

At acidic pH, the vWF D′D3-HSA mutant dimer with the most improved affinity and off rate for CSL627 was EYA with a 500 pM KD and a 10⁻³ l/s off rate. Based on this, the improvement in affinity and off rate for EYA is about 100 fold and at least 10 fold respectively compared to the wild-type dimer.

EYA Dimer appeared to have similar kinetic rates and affinity for CSL627 as S764P/S766I at both neutral and acidic pH. 

1. A modified polypeptide which binds Factor VIII, the modified polypeptide comprising a modified version of the sequence as shown in SEQ ID NO:3 or a fragment thereof in which the modified sequence comprises at least one modification in at least one position selected from the group consisting of L18, V42, K149, N248, S279, V320, T325, Q395, and K418; wherein the modified polypeptide binds to Factor VIII with an off rate lower than a reference polypeptide comprising an unmodified SEQ ID NO:3.
 2. The modified polypeptide of claim 1, wherein the modified polypeptide binds to Factor VIII with an off rate at least 5 fold lower than the reference polypeptide.
 3. The modified polypeptide of claim 1, wherein the modified polypeptide binds to Factor VIII with an off rate at least 10 fold lower than the reference polypeptide.
 4. The modified polypeptide of claim 1, wherein the modified polypeptide binds to Factor VIII with a dissociation constant (KD) at least 5 fold lower than the reference polypeptide.
 5. The modified polypeptide of claim 4, wherein the modified polypeptide binds to Factor VIII with an off rate at least 10 fold lower than the reference polypeptide.
 6. The modified polypeptide of claim 1, wherein the modified polypeptide comprises at least two modifications.
 7. The modified polypeptide of claim 1, wherein the modified polypeptide comprises at least three modifications.
 8. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:5 (S764P/S766W/V1083A).
 9. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:6 (S764G/S766Y/V1083A).
 10. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:7 (S764E/S766Y/V1083A).
 11. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:8 (N1011S/V1083A/K1181E).
 12. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:17 (S766Y/V1083A).
 13. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:9 (V1083A).
 14. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:10 (S1042T).
 15. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:11 (V805A/Q1158L).
 16. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:12 (K912E/T1088S).
 17. The modified polypeptide of claim 1, wherein the modified polypeptide comprises SEQ ID NO:13 (L781P).
 18. A modified polypeptide which binds Factor VIII, the modified polypeptide comprising a modified version of the sequence as shown in SEQ ID NO:3 in which the modified sequence comprises modifications at least at position 320 and at positions 1 and/or 3, wherein the modified polypeptide binds to Factor VIII with an off rate lower than a reference polypeptide comprising an unmodified SEQ ID NO:3.
 19. The modified polypeptide of claim 18, wherein the modified polypeptide comprises modifications at least at positions 1, 3, and 320 of SEQ ID NO:3.
 20. The modified polypeptide of claim 18, wherein the amino acid at position 320 of SEQ ID NO:3 is A.
 21. The modified polypeptide of claim 18, wherein the amino acid at position 3 of SEQ ID NO:3 is selected from the group consisting of Y, I, M, V, F, H, R, and W.
 22. The modified polypeptide of claim 18, wherein the amino acid at position 1 of SEQ ID NO:3 is selected from the group consisting of G, P, V, E, Y, A, and L.
 23. The modified polypeptide of claim 1, wherein the modified polypeptide is modified Von Willebrand Factor (VWF).
 24. The modified polypeptide of claim 1, wherein the modified polypeptide further comprises a half-life extending moiety.
 25. The modified polypeptide of claim 24, wherein the half-life extending moiety is a heterologous amino acid sequence fused to the modified polypeptide.
 26. The modified polypeptide of claim 25, wherein the heterologous amino acid sequence comprises polypeptide selected from the group consisting of immunoglobulin constant regions and portions thereof, transferrin and fragments thereof, the C-terminal peptide of human chorionic gonadotropin, solvated random chains with large hydrodynamic volume known as XTEN, homo-amino acid repeats (HAP), proline-alanine-serine repeats (PAS), albumin, afamin, alpha-fetoprotein, Vitamin D binding protein, polypeptides capable of binding under physiological conditions to albumin or immunoglobulin constant regions, and combinations thereof.
 27. The modified polypeptide of claim 24, wherein the half-life extending moiety is conjugated to the modified polypeptide.
 28. The modified polypeptide of claim 27, wherein the half-life-extending moiety is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acids (PSAs), elastin-like polypeptides, heparosan polymers, hyaluronic acid, albumin binding ligands, and combinations thereof.
 29. The modified polypeptide of claim 25, wherein the heterologous amino acid sequence comprises albumin.
 30. The modified polypeptide of claim 29, wherein the N-terminus of the albumin is fused to the C-terminus of the modified polypeptide, either directly or via a spacer.
 31. The modified polypeptide of claim 30, wherein 1 to 5 amino acids at the natural C-terminus of the modified polypeptide have been deleted.
 32. A complex comprising a Factor VIII molecule and the modified polypeptide of claim
 1. 33.-34. (canceled)
 35. A pharmaceutical composition comprising the modified polypeptide of claim
 1. 36. A method of treating a bleeding disorder, comprising administering to a patient in need thereof a pharmaceutically effective amount of the modified polypeptide of claim
 1. 37. The method of claim 36, wherein the bleeding disorder is von Willebrand's disease (VWD) or hemophilia A. 38.-39. (canceled)
 40. A polynucleotide encoding the modified polypeptide of claim
 1. 41. A plasmid or vector comprising the polynucleotide of claim
 40. 42. The plasmid or vector of claim 41, wherein the plasmid or vector is an expression vector.
 43. A host cell comprising the polynucleotide of claim
 40. 44. A method of producing a polypeptide comprising a modified VWF, comprising: (i) culturing the host cells of claim 43 in a culture medium under conditions such that the polypeptide comprising a modified VWF is expressed; and (ii) optionally recovering the polypeptide comprising a modified VWF from the host cells or from the culture medium.
 45. A method of increasing the half-life of Factor VIII, comprising mixing the Factor VIII with the modified polypeptide of claim
 1. 46. The modified polypeptide of claim 18, wherein the modified polypeptide is modified VWF.
 47. The modified polypeptide of claim 25, wherein the heterologous amino acid sequence comprises an Fc fragment.
 48. The modified polypeptide of claim 27, wherein the half-life-extending moiety is a fatty acid chain. 